摘要
目的构建完整的肺炎支原体粘附分子P30蛋白结构基因原核表达质粒并进行表达。方法用引物修饰法将P30基因中唯一的“UGA”码突变为“UGG”,PCR扩增出P30蛋白编码基因后定向克隆入PET32a(+)质粒,构建PET32a(+)/P30重组体,再转化到大肠杆菌BL21中,用IPTG诱导表达外源基因。结果成功扩增出不含“UGA”码的P30基因;经双向测序结果与GenBank注录的P30基因序列一致;SDS-PAGE显示P30基因在BL21中得到完整表达。结论获得含有完整P30基因的重组克隆株并在大肠杆菌中表达了完整的重组蛋白,为进一步探讨P30基因及其编码产物功能打下了基础。
To construct the prokaryotic expression vector carrying the intact the structural gene of P30 protein in Mycoplasma pneumoniae and to express this gene in E. coli, the only one code ＂UGA＂ was changed to ＂UGG＂ by method of primer modification. The P30 gene was amplified by PCR from genome of M. pneumoniae, and cloned into plasmid pET32a（ ＋ ）. And then, the recombinant plasmid pET32a（ ＋ ）-P30 was transformed into E. coli BL21. With 1mmol/L IPTG induction, the recombinant P30 （rP30） was expressed. The PCR products without ＂UGA＂ code was thus obtained and their sequences were consistent with those of P30 gene in GenBank. Meanwhile, the intact P30 gene could express entirely in BL21 cells as demonstrated by SDS-PAGE. It is concluded that the prokaryotic expression vector pET32a（ ＋ ）-P30 was constructed successfully by the methods described in the present study and the recombinant protein P30 was obtained, that could provide an experimental basis for further studies on the P30 gene and rP30 protein.
出处
《中国人兽共患病杂志》
CSCD
北大核心
2005年第12期1068-1070,共3页
Chinese Journal of Zoonoses
